J. mar. bioI. Ass. U.K. (1967) 47, 23-31Printed in Great Britain

23

DETERMINATION OFNITRATE IN SEA WATER BY CADMIUM-COPPER

REDUCTION TO NITRITE*

By E. D. WOOD!, F. A. J. ARMSTRONG2 AND F. A. RICHARDSl

1 Department of Oceanography, University of Washington, Seattle2 The Plymouth Laboratory, England

(Text-figs. 1-2)

An accurate, dependable determination of 0-60 ,ug-at./l. of NOs -N in sea waterhas been developed. The sample is treated with tetrasodium ethylenediamine­tetraacetate solution and passed through a column of copperized cadmiumfilings. A nearly quantitative reduction of nitrate to nitrite results. Nitrite is thendetermined by a diazotization method. Neither sulphide nor high nitrite con­centrations interferes. Approximately eight samples per hour per column can beanalysed with a standard deviation of 0'12 ,ug-at./l. at the 20 ,ug-at./l. level.

INTRODUCTION

Accurate determinations of nitrate ions in sea water have been difficult,especially under shipboard conditions.

The colorimetric method described by Harvey (1926, 1930) and improvedby Cooper (1932), Zwicker & Robinson (1944), and others uses strychnidinein concentrated sulphuric acid to produce a red colour. The reagent lacksreliable sensitivity, because it is dependent on the rates of mixing and cooling.

In a method by Armstrong (1963), the absorbance of nitrosyl chloride inthe UV region is measured with a spectrophotometer. While the method isgood for small samples containing high concentrations of nitrate, the use ofconcentrated sulphuric acid and lack of sensitivity limit its use in routineanalysis.

A method in which nitrate is quantitatively reduced to nitrite would beadvantageous, because nitrite can be readily determined by the sensitivediazotization method proposed by Griess (1879). Several such methods havebeen proposed. F0yn (1951), Vatova (1956), and Chow & Johnstone (1962)used zinc powder for the reduction, but the reduction is sensitive to tempera­ture, and it is necessary to centrifuge or filter each sample. The reduction is notvery reproducible and only 85-90 % of the nitrate is reduced to nitrite (Morris& Riley, 1963).

* Contribution no. 392,Department of Oceanography,University of Washington, Seattle.Reference M66-28.

24 E. D. WOOD, F. A. J. ARMSTRONG AND F. A. RICHARDS

Mullin & Riley (1955) used hydrazine, in the presence of cupric ion as acatalyst, to reduce nitrate to nitrite. This method, as modified by Austin &Strickland (1959), requires 24 h at constant temperature for the reduction andis erratic, especially when used at sea, and unreliable in the presence of highnitrite concentrations (Morris & Riley, 1963).

The use of cadmium as the reducing agent was suggested by Potzl & Reiter(1960) and has proved to have advantages in the routine analysis of nitratein sea water. The method of Morris & Riley (1963) reproducibly reduces9I ± I % of the nitrate to nitrite when samples are percolated through a columnof amalgamated cadmium filings. The method has negligible salt error,minimal temperature effect, and there are no interfering ions in open seawater. The column decreases in efficiency with use because of the formationof Cd(OH)2 (and possibly CdC03).

Grasshoff (1964) modified the method of Morris & Riley (1963) bylengthening the reducing column and treating the metal with an alkalineammonium chloride solution to complex the oxidized CdH ion, thusprolonging the life of the column. The procedure was further refined byStrickland & Parsons (1965, pp. 73-8) by replacing the ammonium chloridewith the tetrasodium salt of ethylenediaminetetraacetic acid, EDT A, for thecolumn conditioner. They also suggested washing the cadmium filings withnitric acid before amalgamation. This appears to pit the Cd, providing greatersurface area.

The method described below uses copper as the cathode rather thanmercury in the reproducible, nearly quantitative reduction of nitrate tonitrite. EDT A is used to complex the cadmium ion. The determination ofnitrite is made with reagents suggested by Shinn (1941). The nitrite ion isdiazotized with sulphanilamide, then coupled with N-(1-naphthyl)-ethyl­enediamine to produce a deep red colour, as described by Bendschneider &Robinson (1952).

APPARATUS

The column (Fig. I) is like that described by Morris & Riley (1963), in that thedischarge tip and the top of the cadmium filling are at the same height, which preventsthe column from running dry. This precaution is essential, because exposure to airquickly decreases the efficiency of the column.

A Teflon stopcock equipped with a metering valve was suggested by Mr NathanielCorwin (personal communication) for better control of the rate of flow.

Best results were obtained using 25 cm of copperized cadmium in a column withan 8 mm bore (Table I). Reduction of the column diameter gave erratic results.

An arbitrary length of about 10 cm between the bottom of the reservoir and thedischarge tip was used to maintain a nearly uniform flow into a graduated container.A stoppered 50 m!. graduated test-tube or stoppered baseless graduated cylinder wasused for each sample.

A compact unit of four columns has been placed on a portable rack on 10-12 cmcentres and used both on shipboard and in the laboratory.

DETERMINATION OF NITRATE 25

I10 em

I

*2 em

mi.

SO

25

30 mml.D.reservoir

Cd-Cu

34 em

ml.SO

40

30

20

10

Stopper

Pyrex glasswool

2 mm capillarytubing

Fig.!. Column used to reduce nitrate to nitrite in sea water, with collectingcylinder. Teflonstopcockwith metering valve (Kimax stopcockno. 4I007-F, It mm bore and capillaryarms).

REAGENTS

Sulphanilamide solution. Dissolve 5 g sulphanilamide in a solution of 50 ml. of12 N-HCl and 300 ml. of distilled water, then dilute to 500 ml. with distilled water.This solution is stable for several months.

26 E. D. WOOD, F. A. J. ARMSTRONG AND F. A. RICHARDS

N-(l-naphthyl)-ethylendiaminedihydrochloride solution. Dissolve 0'50 g of the di­hydrocWoride in distilled water and dilute to 500 m1. This solution is stable for atleast I month if filtered (Whatman no. 42) and then stored in an amber bottle.

Nitrate standard (I mI. = 5,ug-at.). Dissolve 0'1264 g KNOa (dried for I h at110° C) in distilled water and dilute to 250 m1. Add two drops· of cWoroform as apreservative. The solution is stable for many months if stored in a dark, tightly stop­pered glass bottle.

TABLE I. ABSORBANCES OBTAINED VARYING THE Cd-Cu COLUMNLENGTH AND THE CONCENTRATION

(Flow rate = 10 m1./min)Concentrations

Column(,ug-at./litre NO- -N)

length (cm)204060

10

0'4130,8211'15415

0'4620'9171'37920

0'4700'9381'35724

0'4720'9331'36328

0'4710'9361'363

Nitrite standard (I m1. = 5,ug-at.). Dissolve 0'0863 g NaN02 (dried for I h at110° C) in distilled water and dilute to 250 m1. Add two drops of cWoroform as apreservative. This solution is stable for many months when stored in the dark in atightly stoppered glass bottle.

Copper sulphate solution (0'08 M). Dissolve 20 g CuS04.5H20 in 11. of distilledwater.

EDT A solution (0'1 M). Dissolve 38 g of the tetrasodium salt of ethylenediamine-tetraacetic acid in 500 m1. of distilled water and dilute to I 1.

HCl(2 N). Dilute 85 m1. of 12 N-HCI to 500 m1. with distilled water.HNOa (0'3 N). Dilute 10 m1. of 15'4 N-HNOa to 500 mI. with distilled water.HCl (0'0015 N). Dilute 0'125 m1. of 12 N-HCI to I 1. with distilled water.Column wash solution. Add I m1. of the EDT A solution to 50 m1. of 0'0015 N-HC1.

METHODS

Preparation of columns

(I) Coarse cadmium filings are prepared from stick cadmium by placing a stick ina lathe and rasping it with a half-round rasp. The filings are of nearly uniform size,but they may be sifted to obtain the fraction retained on a 0'5 mm screen but passingthrough a 2 mm screen. Drawing a magnet through the filings eliminates most of theiron contamination.

(2) Approximately 40 g per column of the cadmium filings are washed with 2 N-HClin a separatory funnel, then rinsed thorougWy with distilled water. This is followedby washing with 0'3 N-HNOa, rinsing with distilled water, and then washing with2 N-HCl to remove the NO~ ion. A thorough rinsing with distilled water follows.

(3) The cadmium is treated with approximately 100 m1. of the copper sulphatesolution per 40 g of filings in a special washing bottle (Fig. 2). It is well shaken andthen flushed with distilled water, preventing the copperized cadmium from beingexposed to air.

(4) A small plug of Pyrex glass-wool is placed in the bottom of the column, which

DETERMINATION OF NITRATE 27

is then filled with distilled water. The copperized cadmium is introduced slowly byinverting the copperizing vessel into the column reservoir and tapping the columnduring the process to settle the cadmium. (This is important if the column is to be usedaboard ship or subjected to vibration, because the cadmium will settle during use).Do not fill the column above the level of the discharge tip, a safety feature to preventthe column from running dry.

(5) Some copper and fine cadmium settle out on top of the cadmium column andare removed by suction. A small Pyrex glass-wool plug is placed on top of the cadmiumto act as a filter and is replaced when necessary.

Fig. 2. Container used to introduce copperized cadmium to the column without exposure toair by inverting into the top of the column (250 m!. Erlenmeyer flask with rubber stopperholding 8 mm. I.D. glass funnel delivery tube and 2 mm I.D. vent tube).

(6) The cadmium column is washed with approximately 50 mI. of the washsolution. The columns are allowed to stand at least 24 h before use, renewing the washsolution three or four times during this period.

(7) A freshly prepared column reduces approximately 15 % of the nitrate to oxida­tion states lower than that of nitrite. Passing 3-4 1. of water containing approximately60 p,g-at./I. of NO; -N and 20 mI. of EDT A solution/I. through a new columnconditions it sufficiently to yield a nearly uniform 98 % reduction to nitrite.

(8) A column length of 15 cm of Cd-Cu and a flow rate of 10-12 ml./min can beused for determining nitrate in fresh water.

0'4720'4820'4780'4690'413

28 E. D. WOOD, F. A. J. ARMSTRONG AND F. A. RICHARDS

Procedure

(I) One ml. of the EDT A solution is added to 50 ml. of sample in a 50 ml. graduatedcylinder and shaken. Turbid samples must be filtered before treatment.

(2) Any excess sample or wash solution is removed from the column reservoir withsuction to about 0'5 cm above the cadmium. A 3-4 ml. portion of the sample is addedto the column reservoir to rinse out the previous sample: 1-2 ml. are allowed to drainthrough the column before removing the remainder with suction. The remainder ofthe sample is then added to the reservoir, and the graduated cylinder is placed underthe column discharge tip.

(3) The optimum flow rate, about 6 ml./min (I drop/sec) at the 50 ml.level (Table 2)is set with the metering valve. Several 5 ml. portions of the sample are passed throughthe column and used to rinse the collecting cylinder until a total of 20 ml. have beendiscarded, then the next 15 ml. of the reduced sample are collected. The 15 ml. sampleis diluted to 30 ml. with distilled water. If nitrate concentrations are less than aboutro p,g-at./l. NO;; -N, 30 ml. of sample should be collected and not diluted.

TABLE 2. ABSORBANCES OBTAINED USING A 25 cm Cd-Cu COLUMN ANDVARYING THE FLOW RATES

(Concentration = 20 #g-at.jl. NO;-N)

Flow rate(ml.jmin)

369

1218

(4) The sample is then treated as a nitrite sample by the method of Bendschneider& Robinson (1952), as follows. Add I ml. of the sulphanilamide solution and shake.Add I ml. of the dihydrochloride solution after 2-8 min. Wait at least ro min for thecolour to develop. The absorbance should be determined within 2 h on a BeckmanDU spectrophotometer (or other suitable instrument) at a wave-length of 543 mp"

slit width of 0'04 mm, in a I cm cell.(5) At the end of a run, rinse the walls of the column reservoir with column wash

solution to prevent the accumulation of salt.

Standardization

The columns achieve a nearly constant reduction of 99 ± I %. However, occasionallya column showed a slight unexplained decrease in efficiency. The reduction efficiency,E, was determined by comparing the corrected absorbance, A" of a nitrite standardwith the corrected absorbance of a reduced nitrate standard of the same concentration,

E = AC•NO,-. (I)Ac,NO,-

Most of the columns we have used have shown a slight increase in efficiency during4 months of use.

Blanks and standards for each column should accompany each day's samples.Blank. 1'00 ml. of the EDT A solution is added to 50 ml. of 0'0015 N-HCI and the

solution mixed and carried through the complete procedure.The measured absorbancein a I cm cell should not exceed o·oro.

DETERMINATION OF NITRATE 29

(2)

Calibration. For four columns, 500 ml. of filtered sea water oflow nitrate concentra­tion are needed. A standard nitrate solution (1'00 ml.) is placed in a 250 ml. graduatedflask and made to the mark with sea water. Distilled water (1'00 ml.) is put ina secondflask and also made to the mark with sea water: 50 ml. portions from each flask aretreated with EDT A and carried through the procedure. The difference in absorbance,for each column, between the samples to which nitrate was and was not added cor­responds to a difference in nitrate concentrations of 20'0 ,ltg-at./l. NOg --N. Since therelationship between absorbance and concentration is linear, a factor, f, may becalculated,

f - 20- A(SW+20) - Asw'

where Ssw +20 is the absorbance of the sample with added nitrate, and Asw is that ofthe sample without added nitrate.

For a 15 ml. reduced sample diluted to 30 ml., f is approximately 42 in I cm cellsand about 2'2 for IO cm cells and an undiluted sample.

Some nitrite is reduced during the process. This amounts to about I % when nitritesamples are run through the column. If high concentrations of nitrite are present,a correction factor of 0'99 should be applied to the nitrite concentration CN02' Thenitrate concentration of the sample is calculated using the equation

CNOg = fAc - 0'99 CN02, (3)

where Ac is the sample absorbance corrected for the blank and f defined in equa­tion 2).

DISCUSSION

In a neutral or basic solution, the reduction of nitrate proceeds accordingto equation (4) (Grasshoff, 1964):

NOg +H20+2e- -+ N02 +20H-,

and the oxidation of cadmium by equation (5):

Cd+t02+H20 -+ Cd(OH)2' (5)

It is suggested that the reduction of nitrate to nitrite in the present methodproceeds according to the equation

NOg + Cd + EDT A-4 + H20 -+ N02 + Cd(EDT A)-2 + 20H-. (6)

The pH of a sea-water sample increases by 0'5 to nearly 1'0 pH units (de­pending on the concentration ofNOg ions) during the reduction, suggestingthe above mechanism.

The minus 4 and minus 2 charges for the EDT A ions are approximate values,because incomplete dissociation takes place. The dissociation of tetrasodiumEDT A increases with pH while the dissociation of the Cd-EDT A compounddecreases with increased pH (Flaschka, 1959, p. 138).

This method is accurate for nitrate concentrations from 0'05 to 60 ltg-at. II.,the concentration range of nitrate normally found in sea water. The

30 E. D. WOOD, F. A. J. ARMSTRONG AND F. A. RICHARDS

concentration levels and standard deviations for ten samples at each levelare:

40'00 ± 0'26 ,ug-at.jl. NOs -N} ( . 11)usmg I em ce s

20'00 ± 0'12 ,ug-at.jl. NOs -N '

1'00 ± 0'04 ,ug-at.jl. NOs -N (using 10 cm cells and no dilution).

The method has been used in the analysis of some 1500 samples at sea.Turbid samples should be well shaken and filtered through membrane

filters (0'45,u pore size) before reduction. Significantly lower values werefound if the sample was allowed to stand, followed by reduction of the cleardecanted water.

A limited number of samples containing up to 60,ug-at.jl. of sulphide­sulphur can be analysed with nearly complete recovery of nitrate (Morris &Riley, 1963). However, repeated use of the columns will inhibit nitratereduction by the formation of CdS. The sulphide ion can be eliminated beforeintroduction to the column by adding cadmium chloride solution in slightexcess of the sulphide concentration.

If sea water is not available, 0'0015 N-HCl is preferable to distilled waterfor standards and blanks, because the pH, after addition of EDT A, is aboutthe same as for sea water, 8'0-8'2, and the reduction equally efficient.

The copperized-cadmium method of nitrate analysis should be easilyadapted for use with the AutoAnalyzer method described by Brewer & Riley(1965). The increased efficiency, small salt effect, and long life of the reducingagent should increase the accuracy and precision of the AutoAnalyzer method.

Sincere gratitude is expressed to Joel Cline, W. W. Broenkow, Ralph Riley, RoyOlund, Fred Palmer, Giilser Giiner, Edwyna von Gal, Eugene E. Collias, andMeredythe Meller for helpful suggestions, technical assistance, and encouragement.

The work was supported in part by Office of Naval Research Contract Nonr­477(37), Project NR 083012.

REFERENCES

ARMSTRONG,F. A. J., 1963. The determination of nitrate in water by ultra-violetspectrophotometry. Analyt. Chem., Vol. 35, pp. 1292-4.

AUSTIN,K. H. & STRICKLAND,J. D. H., 1959. The direct estimation of ammonia insea water with notes on 'reactive' iron, nitrate, and inorganic phosphorus.J. Cons. perm. into Explor. Mer, VoL 24, pp. 446-9.

BENDSCHNEIDER,K. & ROBINSON,R. J., 1952. A new spectrophotometric determinationof nitrite in sea water. J. mar. Res., Vol. II, pp. 87-96.

BREWER,P. G. & RILEY,J. P., 1965. The automatic determination of nitrate in seawater. Deep-Sea Res., Vol. 12, pp. 765-72.

CHOW,T. J. & JOHNSTONE,M. S., 1962. The determination of nitrate in sea water.Analytica chim. acta, Vol. 27, pp. 441-6.

COOPER,L. H. N., 1932. The determination of nitrate in the sea by reduced strychnine.J. mar. bioi. Ass. U.K., Vol. 18, pp. 161-6.

FLASCHKA,H. A., 1959. EDT A Titrations. New York: Pergamon Press. 138 pp.

DETERMINATION OF NITRATE 31

F0YN, E., 1951. Nitrogen determinations in sea-water. FiskDir. Skr., Ser. Havunder­s0k., Bd. 9, No. 14, pp. 1-7.

GRASSHOFF,K., 1964. Zur Bestimmung von Nitrat in Meer- und Trinkwasser.Kieler Meeresforsch., Bd. 20, No. I, pp. 5-11.

GRIESS,P., 1879. Bemerkungen zu der Abandlung der H. N. Weselsky und Benedikt'Uber einige Azoverbindungen'. Ber. dt. chem. Ges., Bd. 12, pp. 426-8.

HARVEY,H. W., 1926. Nitrate in the sea. J. mar. biol. Ass. U.K., Vol. 14, pp. 71-88.HARVEY,H. W., 1930. Nitrate in the sea. II. J. mar. biol. Ass. U.K., Vol. 15, pp. 183­

19°·MORRIS, A. W. & RILEY, J. P., 1963. The determination of nitrate in sea-water.

Analytica chim. acta, Vol. 29, pp. 272-279.MULLIN,J. B. & RILEY,J. P., 1955. The spectrophotometric determination of nitrate

in natural waters with particular reference to sea water. Analytica chim. acta,Vol. 12, pp. 464-80.

POTZL,L. & REITER,R., 1960. Eine einfache Methode zur Bestimmung von Nitrat­Ionen im atmospharischen Niederschlag und in Aerosol-Kondensaten mitAnwendung aufProbleme der Luftelektrizitat wahrend Niederschlagen. Z. AerosolForsch. u. -Ther., Bd. 8, pp. 252-64.

SHINN, M. B., 1941. A colorimetric method for the determination of nitrite. Ind.Bngng Chem., analyt. Bdn, Vol. 13, pp. 33-5.

STRICKLAND,J. D. H. & PARSONS,T. R., 1965. A manual of seawater analysis,2nd ed. revised. Bull. Fish. Res. Bd Can., No. 125, 203 pp.

VATOVA,A., 1956. Elektrophotometrische Nitratbestimmung im Meerwasser mit demPhotometer 'Elko II'. Dt. hydrogr. Z., Bd. 9, pp. 194-8.

ZWICKER,B. M. G. & ROBINSON,R. J., 1944. The photometric determination ofnitrate in sea water with a strychnidine reagent. J. mar. Res., Vol. 5, pp. 214-32.